1. Field of the Invention
The present invention features novel binding molecules and methods of improving the specific binding affinity of binding molecules, which methods do not use X-ray crystallography. For example, in one aspect, the invention features methods of making antibodies with improved specific binding affinity for a polypeptide produced during prothrombin activation. The present invention is useful for a variety of applications including, e.g., producing binding molecules with improved binding affinity; and screening for binding molecules which are in low abundance.
2. Background
Proteins and polypeptides are linear polymers of amino acids that are often referred to as "amino acid residues". Naturally occurring proteins may contain as many as 20 different types of amino acid residues, each of which contains a unique side chain. The primary structure of a protein is determined by the specific sequence of amino acids in the protein.
Proteins and polypeptides generally fold into three-dimensional structures which are determined by interactions between amino acid residues. Examples of such interactions include hydrogen bonding, hydrophobic interactions, van der Waals (VDW) attraction, and electrostatic (ionic) interactions (reviewed in Stryer, L. Biochemistry 3rd Ed. W. H. Freeman and Co., New York (1988), pp. 15-41).
The three-dimensional structure of proteins and polypeptides can be determined in several ways. For example, X-ray crystallography has been used to analyze the structure of proteins, polypeptides, and small molecules (reviewed in Matthews, B. W., The Proteins 3rd Ed., (Academic Press), 3: 404-590 (1977); Van Holde, K. E. (Prentice-Hall, N.J.) (1971), pp. 221-239). Studies of X-ray resolved proteins and polypeptides have revealed .alpha.-helices, parallel and anti-parallel .beta.-sheets, each of which helps to determine secondary and tertiary structure (see Stryer, L., supra).
Many proteins have internal surfaces (directed away from the environment in which the protein is found) and external surfaces (which are in close proximity to the environment). Typically, hydrophobic residues such as, e.g., tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine are found in or near the internal surface of proteins, whereas hydrophilic residues such as aspartate, asparagine, glutamate, glutamine, lysine, arginine, histidine, serine, threonine, glycine, and proline, are usually found on or near the external protein surface. Protein folding is thus dominated by the packing of hydrophobic groups into the protein interior and away from the generally aqueous solvent, thereby favorably increasing solvent entropy. The amino acids alanine, glycine, serine, and threonine are amphipathic to some extent and can be found on both internal and external protein surfaces.
X-ray crystallography has revealed binding sites (clefts) in binding molecules that form specific complexes with other binding molecules. Examples of such complexes include antibody-hapten, antibody-peptide, receptor-ligand, antibody-antibody complexes, and the complexes formed between major histocompatibility (MHC) proteins and presenting peptides (see e.g., Schulze-Gahmen, U. et al. J. Mol. Biol. 234: 1098 (1993); Sharon, J. PNAS (USA) 87:4814 (1990); Stanfield, R. L. et al. Science 248:716 (1990); Denzin, L. K. et al. J. Biol. Chem. 266:14095 (1991); Stryer, L., supra; Stern, L. J. and D. C. Wiley, Cell 68:465 (1992); published PCT Application No. WO 96/94314; and references cited therein). Antibody binding sites have been mutated to study models of antibody-antigen complex formation (Verdaguer, et al. Embo. J. 14:1670 (1995); Strong et al. Biochem. 30:3739 (1991)).
X-ray crystallography has also identified important amino acids called "contact residues" within the specific complexes formed between binding molecules. In general, a contact residue in a first binding molecule helps to stabilize the specific complex by forming a bond with a suitably positioned contact residue in a second binding molecule.
Although X-ray crystallography is a precise technique for detecting contact residues in binding molecules, it is often expensive and time-consuming. In some instances, it can be extremely difficult or impossible to collect crystallographic data such as, e.g., when a binding molecule fails to crystallize properly. In addition, researchers do not always have access to a facility capable of generating X-ray crystallographic data. Accordingly, it is often difficult or impossible to identify contact residues in binding molecules.
Computer-based modeling techniques for identifying contact residues in antibody-antigen or antibody-hapten complexes are known (see, e.g., Bruccoteri, R. E. et al. Nature 335:564 (1988); Near et al. Mol. Immunology 30, 4:369 (1992); Chothia, C. et al. Nature 342:877 (1989); Ruff-Jamison, S. and Glenney, J. R. Prot. Eng. 6:661 1993); del la Paz, P. et al. Embo J. 5, 2:415 (1986); Roberts, S. et al. Nature 328:731 (1987)). However, the modeling techniques have drawbacks. For example, the techniques generally use X-ray resolved antigens, or alternatively, haptens (semi-rigid structures) of known or easily predicted structure. Accordingly, the modeling techniques are often of limited value when the antigen is not a hapten, or if suitable X-ray crystallographic data is unavailable.
Antibodies which bind blood coagulation antigens are known. One blood coagulation antigen is prothrombin: a protein involved in the control of blood clotting. During blood coagulation, prothrombin is activated to thrombin; an event accompanied by the cleavage of a 271 amino acid peptide (the F1.2 peptide) from the amino terminus of prothrombin (see, e.g., Mann, K. G. et al. Ann. Rev. Biochem. 57:915 (1988) and references cited therein).
It would be desirable to have antibodies which specifically bind the F1.2 peptide with high affinity so that prothrombin activation can be detected in a biological sample.
It would also be desirable to have methods of identifying contact residues in binding molecules without using X-ray crystallography. Such methods would be useful in a variety of applications including, e.g., engineering binding molecules with improved binding affinity; and screening for binding molecules which are in low abundance.